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Journal of Virology, August 2005, p. 9588-9596, Vol. 79, No. 15
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.15.9588-9596.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Utilization of Homotypic and Heterotypic Proteins of Vesicular Stomatitis Virus by Defective Interfering Particle Genomes for RNA Replication and Virion Assembly: Implications for the Mechanism of Homologous Viral Interference

Gyoung Nyoun Kim and C. Yong Kang^*

Siebens-Drake Research Institute, The University of Western Ontario, London, Ontario, Canada N6G 2V4

Received 3 February 2005/ Accepted 20 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV_Ind) are capable of interfering with the replication of both homotypic VSV_Ind and heterotypic New Jersey serotype (VSV_NJ) standard virus. In contrast, DI particles from VSV_NJ do not interfere with the replication of VSV_Ind standard virus but do interfere with VSV_NJ replication. The differences in the interfering activities of VSV_Ind DI particles and VSV_NJ DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV_Ind DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV_NJ DI particles could assemble only with homotypic VSV_NJ viral proteins, although the genomic RNAs of VSV_NJ DI particles could be replicated by using heterotypic VSV_Ind N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanism of homologous viral interference mediated by defective interfering (DI) particles has been the subject of investigation for several decades, with the expectation that an understanding of homologous viral interference at the molecular level may lead us to the development of virus-specific, antiviral therapeutic agents. DI particles are subgenomic virus particles, which are generated from the standard virus during undiluted, high-multiplicity passages. DI particles depend on the viral proteins provided by the standard virus for their replication and maturation, yet they interfere with the replication of standard virus in vitro and in vivo. Vesicular stomatitis virus (VSV) has been frequently utilized to study the mechanism of the homologous viral interference, since VSV DI particles are physically separable from the standard virus. We isolated two panhandle-type DI particles from the two different serotypes of VSV, VSV_Ind DI particle (I_DI) from Indiana serotype (26) and VSV_NJ DI particle (NJ_DI) from New Jersey serotype (4), and characterized their genomic structures and interfering activities in our laboratory (4). Among several types of DI particles of VSV, the panhandle-type DI particles are the most commonly isolated. Panhandle-type DI particles contain various lengths of inverse complementary sequences at the 3' and 5' termini of their genome (13, 21). The 3' genomic terminus of the panhandle-type DI particle is exactly the same as the 3' terminus of the plus-strand antigenome of the standard virus. The essential role of the genomic termini for replication and maturation of the panhandle-type DI particles was demonstrated by replacing the internal region with sequences of non-VSV origin in the VSV_Ind DI particle genome (23). The significance of the terminal sequences of panhandle-type DI particle genomes in homologous viral interference has not yet been examined directly.

The VSV genome encodes five proteins that are essential for its transcription, replication, and assembly: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and a large RNA-dependent RNA polymerase (L). Although the mechanism of homologous viral interference has not been illustrated, it is widely believed that VSV DI particles outcompete the standard virus for certain viral proteins, particularly the polymerase complex (comprised of the P and L proteins), during their replication (7). It has been reported that DI particles inhibit the synthesis of full-length genomic RNA of the standard virus rather than primary transcription of mRNAs (9, 10, 14). The transcription of VSV mRNA from the ribonucleoprotein (RNP) complex of one serotype is carried out only by the homotypic polymerase complex, which has been demonstrated by in vitro reconstitution assay (2, 5, 11). However, it has not been determined whether or not the RNA-N protein complex of one serotype can be utilized as a template for RNA replication by the heterotypic polymerase complex. To determine how genomic RNAs and RNPs of DI particles from one serotype can utilize the proteins from the other serotype of VSV, we constructed four different cDNA plasmids encoding chimeric DI RNA genomes representing VSV_Ind [pI_DI(50-50) and pI_DI(255-268)] and VSV_NJ [pNJ_DI(50-50) and pNJ_DI(227-188)]. To understand the molecular mechanism that leads to the different outcomes of the heterotypic interference by VSV_Ind and VSV_NJ DI particles, we investigated the replication and maturation of DI particles in the presence of homotypic and heterotypic VSV proteins using the VSV reverse genetics system. Here we show the difference between VSV_Ind DI particles and VSV_NJ DI particles in terms of the ability to utilize heterotypic proteins for genome replication as well as particle assembly and maturation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmids. Plasmids containing the VSV_Ind DI particle genome with sequences [pI_DI(50-50) and pI_DI(255-268)] and plasmids containing the VSV_NJ DI particle genome (Hazelhurst strain) with sequences [pNJ_DI(50-50) and pNJ_DI(227-188)] were generated by replacing internal sequences of the VSV DI particle genomes with 2,322 bp of bacteriophage sequence as shown in Fig. 1.

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FIG. 1. Schematic representation of chimeric pDIs containing bacteriophage sequences. The internal region of the 2,322-bp HindIII fragment of DNA is shown as a shaded bar. Numbers in the names of the plasmids indicate the length (in nucleotides) of wild-type sequences at the 3' and 5' genomic termini of chimeric DI RNAs. The sizes of chimeric DI RNAs encoded from the plasmids are shown at the right side of the plasmids. Eighty-eight-nucleotide deletions (88 nts) in the DNA of pNJDI(50-50) are shown under the shaded bar. T7P, HDV, and T7T indicate the T7 transcriptional promoter, hepatitis delta virus ribozyme sequences, and T7 transcriptional terminator, respectively. Numbers in the bracket indicate conserved 3' and 5' VSV-specific nucleotide sequences of DI particles.

The cDNA clones of VSV genes from VSV_Ind and the Hazelhurst strain of VSV_NJ were constructed in the pBluescript II KS vector (Stratagene, La Jolla, CA). The clones are nucleocapsid protein N gene clones (pBKS-N_I and pBKS-N_NJ), phosphoprotein P gene clones (pBKS-P_I and pBKS-P_NJ), matrix protein M gene clones (pBKS-M_I and pBKS-M_NJ), and glycoprotein G gene clones (pBKS-G_I and pBKS-G_NJ). The expression of the proteins from the cDNA clones has been confirmed previously in our laboratory (4). We have described construction, expression, and functional analyses of the plasmids encoding L proteins of VSV_NJ (pBKS-L_NJ) recently (15). A plasmid encoding VSV_Ind L (pGem4-L_I) was kindly provided by M. Schubert (National Institutes of Health [NIH], Bethesda, MD). Expression of the VSV proteins from these plasmids was under the control of the T7 transcriptional promoter.

Recovery of chimeric DI particles from cDNA transfection. Chimeric DI particles were recovered from cDNAs by transfection using the calcium phosphate precipitation method and reverse genetics of VSV (16, 24).

Virus, chimeric DI particle preparation, and antibody. A heat-resistant strain of VSV_Ind (originally obtained from L. Prevec, McMaster University, Hamilton, Ontario, Canada) and the Hazelhurst strain of VSV_NJ (a gift from S. Emerson, NIH) were used for the study. Recombinant vaccinia virus, vTF7-3 (6), was a kind gift from Bernard Moss (NIH) and was used as the source of T7 DNA-dependent RNA polymerase in the VSV reverse genetics system. Stock of vTF7-3 was prepared in TK^– 143 cells as described by Mackett et al. (20).

The chimeric DI particles of VSV recovered from cDNA transfection were amplified by coinfecting BHK-21 cells with the corresponding, homotypic VSV standard virus at a multiplicity of infection (MOI) of 3. The amplified chimeric DI particles were purified by a gradient centrifugation using 5% to 30% linear sucrose gradients prepared in TNE buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA). The collected chimeric DI particles were stored at –80°C. To estimate DI particle concentration, the total protein of the DI particle was quantified with a protein assay kit (Bio-Rad, Hercules, CA).

Polyclonal rabbit antibodies against total proteins of VSV_Ind and VSV_NJ were prepared previously in our laboratory using sucrose gradient-purified and lysed whole viruses (4).

Analysis of RNA synthesis. For the analysis of chimeric DI RNA replication in the presence of the N, P, and L proteins of VSV, one of the chimeric pDIs encoding the positive-strand antigenomic RNA of chimeric DI and plasmids encoding N, P, and L of VSV_Ind or VSV_NJ were transfected into BHK-21 cells, which were preinfected with recombinant vaccinia virus vTF7-3, at an MOI of 5. Transfection was carried out using Lipofectin (Invitrogen, Carlsbad, CA) according to manufacturer's protocol. The concentrations of the plasmids used for the transfection were 1.4 pmol (5 to 5.5 µg) of chimeric pDIs and 6 µg, 3 µg, and 1 µg of pN, pP, and pL, respectively. Chimeric DI RNA-N protein complexes were isolated from cell lysates prepared at 30 h posttransfection by immunoprecipitation. Immunoprecipitation of RNA-N protein complexes and RNA extraction were done as described previously (15). The level of chimeric DI RNA synthesis was determined by Northern blot analysis using sequence-specific riboprobes -E/E1 and -E/E2 (15). The RNA bands were detected by autoradiography, and the intensity of the bands was measured using a densitometer (ImageMaster; Amersham-Pharmacia, Piscataway, NJ).

Genomic RNA and mRNA synthesis of the standard virus and genomic RNA synthesis of the chimeric DI particles in cells coinfected with the standard virus were examined by labeling infected cells with [³H]uridine (30 µCi/ml) (NEN, Boston, MA) in the presence of 10 µg/ml actinomycin D. The cells were radiolabeled for 2 h at 4 h postinfection. RNAs were isolated from the cells and analyzed by electrophoresis in a 1% agarose formaldehyde gel (3). After the completion of gel electrophoresis, the gel was fixed with a solution containing 30% methanol and 10% acetic acid for 60 min. The gel was washed twice with methanol for 30 min each and then treated with a 3% solution of 2,5-diphenyl-oxazole (Sigma-Aldrich, St. Louis, MO) for 3 h. The gel was rehydrated in distilled H₂O for 60 min and dried using a gel drier (Bio-Rad). The RNA bands were detected by autoradiography.

Interference assay. The interfering activities of purified DI particles were examined by the standard virus yield reduction assay as described previously (26, 27, 31).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Usage of P and L proteins by RNA-N protein complex of DI particles. The functional template for the replication of the DI particle is the genomic RNA encapsidated with N protein. It has been demonstrated that the polymerase complex (P and L proteins) of isolated RNP can be interchanged with the added P and L proteins (8). It may be possible that the capability of RNPs of DI particles from one serotype to utilize P and L proteins provided by the standard VSV of the other serotype determines the heterotypic interfering activities of DI particles. Therefore, we examined the utilization of P and L proteins of both VSV_Ind and VSV_NJ by DI genomic RNA-N protein complex from both serotypes of VSV. We analyzed the synthesis of two of the DI RNAs [I_DI(50-50) and NJ_DI(50-50)] (Fig. 1) in various combinations of N, P, and L proteins from the two different serotypes. The detection of RNA synthesis by Northern blot analysis is depicted in Fig. 2. Figure 2B shows the replication of I_DI(50-50) in the presence of different combinations of N, P, and L proteins from VSV_Ind and VSV_NJ. T7 RNA polymerase-mediated positive-strand RNA synthesis and encapsidation were detected in all combinations (Fig. 2B, open block arrow); however, VSV RNA polymerase-derived negative-strand RNA synthesis was detected with only three combinations of N, P, and L proteins; (i) N_Ind, P_Ind, and L_Ind; (ii) N_NJ, P_NJ, and L_NJ; and (iii) N_NJ, P_Ind, and L_NJ (Fig. 2B, lanes 2, 4, and 10). NJ_DI(50-50) replicated in the presence of the same combinations of N, P, and L proteins as I_DI(50-50) (Fig. 2C), although the efficiency of replication in the presence of N_Ind, P_Ind, and L_Ind was lower than that in the presence of N_NJ, P_NJ, and L_NJ. Synthesis of NJ_DI(50-50) RNA in the presence of N_NJ, P_Ind, and L_NJ was also detected, indicating that not only I_DI(50-50) but also NJ_DI(50-50) can be replicated by the combination of N_NJ, P_Ind, and L_NJ proteins. These results demonstrate that N, P, and L proteins of one serotype do not exclude the genomic RNAs of VSV DI particles as long as all three proteins are from the same serotype or three proteins are in the combination of N_NJ, P_Ind, and L_NJ. The results also demonstrate that DI RNP complex from one serotype does not utilize P and/or L proteins from the other serotype. It was interesting to see that replication of chimeric DIs was carried out by the combination of N_NJ, P_Ind, and L_NJ but not with the combination of N_Ind, P_NJ, and L_Ind. These results clearly demonstrate that P_Ind can interact with N and L proteins from both serotypes of VSV. In contrast, P_NJ can interact only with N and L proteins of VSV_NJ.

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FIG. 2. Replication of IDI(50-50) and NJDI(50-50) RNAs with combinations of N, P, and L proteins from VSVInd and VSVNJ. The encapsidated positive-sense and negative-sense chimeric DI RNAs were detected by Northern blot analysis. (A) Schematic representation of DI RNA synthesis after transfection. -E/E1 and -E/E2 represent the riboprobes for the detection of RNA synthesis by Northern blot analysis. (B) Replication of IDI(50-50) RNA with combinations of N, P, and L proteins from VSVInd and VSVNJ. (C) Replication of NJDI(50-50) RNA with combinations of N, P, and L proteins from VSVInd and VSVNJ. Combinations of N, P, and L proteins from VSVInd and VSVNJ are shown in the boxes. The open arrows indicate the RNA synthesized by T7 RNA polymerase and encapsidated by N proteins. The RNA-N protein complexes were immunoprecipitated with anti-VSV sera. I and NJ denote proteins from VSVInd and VSVNJ, respectively.

Usage of homotypic and heterotypic N, P, and L proteins by DI particle genomic RNA for RNA replication. The genomic RNAs of DI particles from both serotypes containing only 50 nucleotides of terminal sequences can utilize N, P, and L proteins as long as all three proteins are from the same serotypes (Fig. 2). The panhandle-type DI particles contain cis-acting promoter sequences at the 3' termini of both positive- and negative-strand genomes. It has been previously demonstrated that the VSV_Ind DI particle genome requires only 45 nucleotides of 3' and 5' genomic terminal sequences to replicate efficiently (18). The minimal number of terminal nucleotides required for efficient replication of VSV_NJ DI particles has not been determined previously. The NJ_DI genome contains 71-nucleotide-long inverse complementary sequences (15), which are longer than that of I_DI (54 nucleotides). We compared the genomic RNA replication between DI particles containing 50 nucleotides of genomic end sequences, I_DI(50-50) and NJ_DI(50-50), and DI particles containing more than 50 nucleotides of genomic end sequences, I_DI(255-268) and NJ_DI(227-188). To determine the replication efficiency of DI RNAs using homotypic and heterotypic N, P, and L proteins, DI RNA synthesis was analyzed in the presence of N, P, and L proteins from either VSV_Ind or VSV_NJ as depicted in Fig. 2. The amounts of encapsidated plus-strand RNA synthesized from the chimeric pDI by T7 RNA polymerase were similar among the chimeric DI particles (Fig. 3, open block arrow), indicating that approximately equal numbers of templates were available for negative-strand RNA synthesis by the VSV RNA polymerase. All chimeric DI RNAs were synthesized by the VSV_Ind N, P, and L proteins with differing efficiencies. Although chimeric VSV_NJ DI RNAs were synthesized by the heterotypic VSV_Ind N, P, and L proteins, the amount of chimeric VSV_NJ DI RNA synthesis was significantly lower than that of chimeric VSV_Ind DIs (Fig. 3A). In contrast, the N, P, and L proteins of VSV_NJ supported replication of chimeric I_DI(50-50) and I_DI(255-268) as efficiently as its homotypic NJ_DI(227-188) particle (Fig. 3B). Our results demonstrate that genomic RNAs of DI particles can utilize N, P, and L proteins from either serotype, but only I_DI RNA can replicate with high efficiency using N, P, and L proteins from both serotypes.

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FIG. 3. Replication of chimeric DI RNAs in the presence of homotypic and heterotypic N, P, and L proteins. Open block arrows indicate the chimeric DI RNAs synthesized by T7 RNA polymerase and encapsidated with N proteins. VSV polymerase-specific (second strand) RNA synthesis of chimeric DI RNAs in the presence of homotypic and heterotypic N, P, and L proteins is shown in the left panels. Average values relative to IDI(255-268) and NJDI(227-188) from three experiments are shown in the graphs. Error bars represent standard deviations of the mean. (A) Replication of chimeric DI RNAs in the cells expressing VSVInd N, P, and L proteins. (B) Replication of chimeric DI RNAs in the cells expressing VSVNJ N, P, and L proteins.

In the presence of VSV_Ind N, P, and L proteins, similar amounts of negative-strand RNAs were synthesized from I_DI(50-50) and I_DI(255-188), indicating that 50 nucleotides of I_DI at both the 3' and 5' genomic termini are sufficient for replication and encapsidation (Fig. 3A, lanes 2 and 3). In contrast, NJ_DI(50-50) particles were only 30% as efficient in the synthesis of negative-strand RNA as NJ_DI(227-188) (Fig. 3B, lanes 3 and 4). These results suggest that the VSV_NJ DI particles require more than 50 nucleotides of terminal genomic sequences for the optimum level of genomic RNA replication.

Assembly of DI particles using homotypic and heterotypic proteins. Assembly and maturation of VSV is mediated by the interaction of RNP complexes with matrix proteins (M) and glycoproteins (G) (19, 22, 34). VSV_Ind DI particles are able to replicate and mature in cells coinfected with either VSV_NJ or VSV_Ind standard virus (26, 28). On the other hand, VSV_NJ DI particles are able to replicate and mature only in cells coinfected with the homotypic VSV_NJ standard virus. It is now clear that genomic RNAs of VSV_NJ DI particles can be encapsidated by the N protein of VSV_Ind and can be associated with the polymerase complex (P and L) of VSV_Ind to form a functional RNP complex (Fig. 3A). Therefore, we decided to examine whether or not genomic RNA of the VSV_NJ DI particle could be assembled into DI particles in the presence of N, P, L, M, and G proteins of VSV_Ind. Recovery of VSV_Ind DI particles from cDNA was also performed with the same set of proteins using the reverse genetics of VSV (16, 24). In the presence of N, P, L, M, and G proteins of VSV_Ind, only VSV_Ind DI particles were recovered from transfected cells and amplified by coinfection with standard VSV_Ind or VSV_NJ (Fig. 4, lanes 1 and 2). We were unable to recover VSV_NJ DI particles in the presence of VSV_Ind proteins (Fig. 4, lanes 3 and 4), indicating that VSV_NJ DI particles cannot assemble using VSV_Ind proteins although their genomic RNAs can be replicated in the presence of N, P, and L proteins of VSV_Ind. In contrast, all chimeric DIs [NJ_DI(50-50), NJ_DI(227-188), I_DI(50-50), and I_DI(255-268)] were recovered from cDNAs when all five VSV_NJ proteins were coexpressed (Fig. 4, lanes 6, 7, and 8). The amount of I_DI(50-50) particle recovered was extremely low, necessitating a longer exposure to see a band on the Northern blot. These findings demonstrate that the I_DI genome replicates and assembles using proteins from VSV_NJ, as expected. However, genomes of NJ_DI particles cannot be assembled using VSV_Ind proteins, despite the fact that the genomes of these DI particles can be replicated at a low level in the presence of N, P, and L proteins of VSV_Ind. We also found that I_DI(50-50) and NJ_DI(50-50) matured poorly with VSV_NJ proteins. These results suggest that the VSV_Ind proteins can assemble only with VSV_Ind genomes and that an appropriate length of genomic terminal sequences is required in the assembly of VSV_NJ and VSV_Ind virions.

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FIG. 4. Assembly and maturation of chimeric DI particles using homotypic and heterotypic VSV proteins. The recovered chimeric IDI and NJDI particles from the transfections were concentrated by ultracentrifugation and were amplified by coinfecting BHK-21 cells with either VSVInd or VSVNJ standard viruses at an MOI of 3. VSVInd was used as a helper virus for the DI particles recovered by using VSVInd N, P, M, G, and L proteins. VSVNJ was used as a helper virus for the DI particles recovered by using VSVNJ N, P, M, G, and L proteins. The presence of chimeric DI particles in the culture fluid was determined by infecting fresh BHK-21 cells with the culture fluid from the first amplification and Northern blot analysis using the sequence-specific riboprobe -E/E2.

Genomic terminal sequences of DI particles confer interfering activity. We examined the interfering activities of purified I_DI(50-50) and NJ_DI(227-188) against the homotypic and heterotypic standard viruses to determine the role of genomic termini in homologous viral interference. BHK-21 cells were infected at an MOI of 3 with standard VSV (VSV_Ind or VSV_NJ) and superinfected with various concentrations (16 ng, 64 ng, 256 ng, and 1,024 ng) of DI particles. At 18 h after infection, culture medium was harvested and the titer of the standard virus was measured by plaque assay. I_DI(50-50) interfered with the replication of both VSV_Ind and VSV_NJ. The yield reduction of the standard virus was correlated with the increased amount of input DI particles in both homotypic and heterotypic viral interference assays (Fig. 5). Although I_DI particles could interfere heterotypically with the replication of VSV_NJ, 6- to 20-fold more I_DI(50-50) particles were required to achieve the same level of interference as with the homotypic standard virus (Fig. 5A). NJ_DI(227-188) particles also showed the same level of interfering activity as their homotypic wild-type VSV_NJ DI particles (data not shown). These VSV_NJ DI particles interfered with the replication of the homotypic VSV_NJ standard virus, resulting in a standard virus yield from 300- to 1,000-fold less at the highest concentrations of input DI particles. However, these VSV_NJ DI particles did not interfere with the replication of the heterotypic VSV_Ind standard virus, even with the highest concentration (Fig. 5B). These results demonstrate that the interfering activity of the panhandle-type DI particles of VSV was conferred by the 3' and 5' genomic termini, which contain promoter sequences, but not by the internal region of the DI particle genome.

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FIG. 5. Interfering activity of chimeric DI particles. The yields of the standard viruses represent the mean value of duplicate plaque assays. (A) Yield reduction of VSV standard viruses by IDI(50-50). (B) Yield reduction of VSV standard viruses by NJDI(227-188). Error bars represent the standard deviation of the mean of three separate assays.

In cells coinfected with VSV DI particles and the standard virus, DI particles inhibit the replication of full-length standard virus genomic RNA and secondary transcription of the mRNAs, while genomic RNA of DI particles is synthesized preferentially to a certain saturation point (4). Therefore, we examined the synthesis of standard virus genomic RNA and genomic RNA of DI particles in cells infected with both standard virus and chimeric DI particles. BHK-21 cells were infected with an MOI of 3 of VSV_Ind or VSV_NJ and superinfected with various concentrations of I_DI(50-50) or NJ_DI(227-188) (1 ng, 4 ng, and 16 ng for the homotypic standard virus and 64 ng, 240 ng, and 960 ng for the heterotypic standard virus). Cells were labeled with [³H]uridine (30 µCi/ml) for 2 h. Total cellular RNAs were isolated and analyzed in a 1% agarose formaldehyde gel. The RNA bands were visualized by autoradiography. I_DI(50-50) showed a dramatic inhibition of VSV_Ind genomic RNA and mRNA synthesis with increasing amounts of DI particles (Fig. 6). The synthesis of I_DI(50-50) genomic RNA increased gradually with larger amounts of input DI particles (Fig. 6A). I_DI(50-50) inhibited genomic RNA and mRNA synthesis of VSV_NJ as well, but much greater amounts of DI particles were required to obtain the same level of interfering activity compared to the VSV_Ind RNA synthesis (Fig. 6A). The amount of I_DI(50-50) genomic RNA synthesized with the help of VSV_NJ was not as large as that of I_DI(50-50) genomic RNA synthesized in the presence of homotypic VSV_Ind. Similar levels of I_DI(50-50) genomic RNA were synthesized in cells coinfected with 240 ng and 960 ng of the DI particles and the VSV_NJ standard virus (Fig. 6A, lanes 7 and 8). NJ_DI(227-188) inhibited genomic RNA and mRNA synthesis of VSV_NJ in a gradual manner with increasing concentration of the DI particles (Fig. 6B). In contrast, NJ_DI(227-188) did not affect the genomic RNA and mRNA synthesis of VSV_Ind, although we used as much as 960 ng per culture dish (Fig. 6B).

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FIG. 6. Analysis of VSV RNA synthesized in cells coinfected with chimeric DI particles and the standard viruses. (A) Synthesis of IDI(50-50) genomic RNA and genomic RNA and mRNA of VSVInd and VSVNJ standard virus in the presence of various concentrations of input IDI(50-50). (B) Synthesis of genomic RNA of NJDI(227-188) and genomic RNA and mRNA of VSVInd and VSVNJ with various concentrations of NJDI(227-188). Arrows indicate the genomic RNAs of the chimeric DIs. Std, standard virus; L, G, N, and M/P, mRNA of each gene.

To our surprise, a small amount of NJ_DI(227-188) RNA was replicated with the help of the VSV_Ind standard virus (Fig. 6B). We have confirmed that the low level of NJ_DI(227-188) RNA synthesis was not the result of copurified VSV_NJ standard virus in the DI particle preparation (data not shown). The result was confirmed by Northern blot analysis using the sequence-specific riboprobe, which is much more sensitive and detects internal sequences in the NJ_DI(227-188) RNA directly (data not shown). These results show that VSV_NJ DI particles can replicate at a very low efficiency with the help of the VSV_Ind standard virus, although NJ_DI failed to interfere heterotypically.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Both standard VSV and DI particles incorporate all five VSV proteins during assembly and maturation (1, 12, 32). Although panhandle-type DI particles cannot produce any VSV proteins, it is assumed that they can initiate their genomic RNA synthesis by using the polymerase complex incorporated into the DI particles (30). If DI particles are to interfere with the replication of the standard virus homotypically and/or heterotypically, the DI particle RNPs or genomic RNAs synthesized from the initial templates should be able to utilize the proteins from the standard virus efficiently. Because only the genomic RNAs encapsidated with N protein as a form of RNP complex function as templates for transcription and replication, it has been difficult to demonstrate in vitro whether or not the DI genomic RNA of one serotype can utilize the proteins of the other serotype. It has been demonstrated that VSV_Ind DI particles could utilize proteins from the VSV_NJ standard virus for their replication in infected cells (28). However, genomic RNA synthesis of the VSV_NJ DI particle has not been demonstrated in cells coinfected with the VSV_Ind standard virus. It has also been demonstrated that P and L proteins in the RNP are interchangeable with the newly added P and L proteins in vitro (8). Therefore, we expected that the RNP complex of VSV_Ind DI particles could exchange with P and L proteins of VSV_NJ but that the RNP complex of VSV_NJ DI particles could not do the reverse. DI particle genomic RNAs of both VSV_Ind and VSV_NJ replicated when N, P, and L proteins were from the same serotype or a combination of N_NJ, P_Ind, and L_NJ (Fig. 2B and C). Any other combinations of N, P, and L proteins did not support the replication of VSV_Ind DI particle or VSV_NJ DI particle genomes. Combinations of N_Ind, P_Ind, and L_NJ or N_Ind, P_NJ, and L_NJ did not support the replication of the I_DI genome, demonstrating that the RNA-N protein template of the VSV_Ind DI particle or the VSV_NJ DI particle cannot utilize the P and L proteins of the heterotypic standard virus directly. Overall, the data shown in Fig. 2 demonstrate that protein interactions among RNA-N protein complexes, P proteins, and L proteins play a crucial role in conferring serotypic specificity of the VSV.

It was not known previously whether or not the genomic RNAs of VSV_NJ could utilize VSV_Ind proteins for their replication. Our results shown in Fig. 3 demonstrate that genomic RNAs of VSV_NJ DI particles utilized N, P, and L proteins of VSV_Ind for their replication. However, the efficiency of the VSV_NJ DI particle RNA replication in the presence of VSV_Ind N, P, and L proteins was much lower. This shows that DI particle genomic RNAs from both serotypes can utilize VSV proteins without serotypic exclusion but with differing efficiencies.

It has previously been demonstrated that panhandle-type VSV_Ind DI particle genomes containing 45 nucleotides of the 3' and 5' terminal genomic sequences replicated efficiently in the presence of homotypic proteins (23). Our data in Fig. 3 agreed with the previous results by showing that I_DI(50-50) and I_DI(255-268) replicated equally well with both homotypic and heterotypic N, P, and L proteins. VSV_NJ DI particle genomes containing only 50 nucleotides of terminal genomic sequences [NJ_DI(50-50)] were less efficient in their replication than DI particle genomes containing more than 188 nucleotides of the VSV_NJ-specific terminal genomic sequences [NJ_DI(227-188)]. Considering the long 3' and 5' inverse complementary sequence of the NJ_DI particle genome (71 bases), it may be possible that VSV_NJ DI particles containing longer inverse complementary sequences in their genomes are generated from the standard virus and are selectively amplified because of the advantage they have in utilizing the polymerase complex or other viral proteins involved in the assembly of the VSV virions.

VSV virion matures by budding through the cytoplasmic membrane as a result of the specific interaction of the RNP complex with the matrix protein (M) and glycoprotein (G) (17, 22). Since chimeric VSV_NJ DI RNAs can replicate using the N, P, and L proteins of VSV_Ind (Fig. 3A), which suggests the assembly of functional RNP, they should be able to mature and bud out as DI particles if M and G proteins of VSV_Ind are provided. However, the results in Fig. 4 show that the maturation of VSV_NJ DI particles using VSV_Ind proteins does not occur. These data can be interpreted in one of two ways. The first, a very low level of replication of NJ_DI genome using N, P, and L proteins of VSV_Ind results in few RNA-N protein complexes which are available for assembly by M and G proteins of VSV_Ind. The second is that specific genomic terminal sequences of DI particles may be involved in the maturation of VSV particles as described by others (33).

Pattnaik et al. have shown that chimeric VSV_Ind DI particles containing 51 nucleotides from the 3' and 5' genomic termini of a panhandle-type DI particle with non-VSV internal sequences could replicate and mature into infectious DI particles (23). These investigators examined the maturation of the chimeric DI particle without analyzing the interfering activity of the chimeric DI particles. We successfully recovered, amplified, and purified I_DI(50-50), I_DI(255-268), and NJ_DI(227-188) particles using the homotypic standard viruses (Fig. 4, lanes 1, 2, and 8). The interfering activities of the recovered chimeric DI particles were the same as those of wild-type DI particles, indicating that genomic terminal sequences encompassing the promoter sequences in panhandle-type DI particle genomes are the only elements required for interfering activity.

We propose the following model of homotypic and heterotypic viral interference mediated by VSV DI particles. In the case of homotypic viral interference, RNPs of VSV DI particles utilize their own as well as newly synthesized polymerase complexes provided by the standard virus to initiate antigenomic RNA synthesis. Once the RNAs are synthesized from the RNP of DI particles, the nascent RNA genomes are encapsidated by N proteins synthesized by the standard virus and form new RNPs. While DI particle genomic RNA replicates using N, P, and L proteins without the need for transcription, standard viruses have to transcribe mRNAs to provide the viral proteins necessary for replication. Because its 3' and 5' inverse complementarity of panhandle-type DI particle genome provides stronger promoter sequences to both senses of the DI particle genome (18, 23) and the DI particle is dependent on the standard virus for the N, P, and L proteins (25), DI particles have a replicative advantage over the standard virus. Consequently, DI particles interfere with the replication of the standard virus.

In the case of heterotypic interference, when cells are coinfected with DI particles and a heterotypic standard virus, DI particle RNA-N protein complexes from either serotype cannot utilize P and L proteins from different serotypes (Fig. 2). They require initial transcription of leader RNAs through the use of their own DI particle-associated P and L proteins. The incapability of I_DI RNPs to directly use VSV_NJ P and L proteins may delay the replication of the DI particle genome. The delay of I_DI particle replication may give the VSV_NJ standard virus a chance to initiate replication using the available N, P, and L proteins and subsequently results in the reduced interfering activity against the VSV_NJ standard virus. This possibility is supported by an earlier observation that adding DI particles at later times of standard virus infection fails to inhibit the genomic RNA and mRNA synthesis of the standard virus (29). The efficiency of the promoter of the VSV_NJ DI particle genome using the polymerase complex from VSV_Ind would not be sufficient to compete for VSV_Ind P and L proteins (Fig. 3). Therefore, VSV_NJ DI particles cannot interfere with the replication of VSV_Ind standard virus, although genomic RNAs of VSV_NJ DI particles can be replicated by N, P, and L proteins of VSV_Ind with very low efficiency (Fig. 3).

Another difference between the VSV_Ind and VSV_NJ DI particles in using heterotypic proteins is the inability of VSV_NJ DI particles to assemble into virions using VSV_Ind proteins (Fig. 4). Although we cannot directly demonstrate whether or not the ability to assemble using heterotypic proteins contributes to heterotypic interference by VSV_Ind DI particles, we speculate that the assembly of VSV_Ind DI particles using VSV_NJ proteins will amplify the DI particles and the amplified DI particles will spread to neighboring cells, which will result in a reduction of the standard virus yield. In contrast to the VSV_Ind DI particles, the VSV_NJ DI particles cannot assemble using heterotypic VSV_Ind proteins, although their genomic RNA can be replicated by VSV_Ind proteins at a low level; therefore, the input DI particles are restricted to the cells initially infected. Our results suggest that both DI particle genomic RNA replication and particle assembly are prerequisites for efficient homologous viral interference. In addition, the ability of the genomic RNAs of DI particles to replicate and mature as DI particles efficiently using heterotypic VSV proteins allows DI particles to interfere heterotypically.

 
We thank Chad Michalski and Rosanne Kang for correcting the manuscript.

This study was supported by grants from the Canadian Institute of Health Research (CIHR) and Sumagen Company Limited.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Siebens-Drake Research Institute, The University of Western Ontario, 1400 Western Road, London, Ontario N6G 2V4, Canada. Phone: (519) 661-3226. Fax: (519) 661-3359. E-mail: cykang{at}uwo.ca.

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Journal of Virology, August 2005, p. 9588-9596, Vol. 79, No. 15
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.15.9588-9596.2005
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